Mycobiology
Research Note
Three New Records of Penicillium Species
Isolated from Insect Specimens in Korea
1
1
1
1
1
2
Kabir Lamsal , Sang Woo Kim , Shahram Naeimi , Mahesh Adhikari , Dil Raj Yadav , Changmu Kim ,
3
1,
Hyang Burm Lee and Youn Su Lee *
1
Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-701, Korea
Microorganism Resources Division, National Institute of Biological Resources, Incheon 404-708, Korea
3
Division of Applied Bioscience and Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju
500-757, Korea
2
Abstract Three Penicillium species have been isolated from insect specimens in Korea; Penicillium sp., P. steckii, and P.
polonicum. Penicillium sp. (KNU12-3-2) was isolated from Lixus imperessiventris, while P. polonicum (KNU12-1-8) and Penicillium
steckii (KNU12-2-9) were isolated from Muljarus japonicas and Meloe proscarabaeus, respectively. The identification was based on
the morphological characteristics of the fungi and in internal transcribed spacer analysis. This is the first report on the isolation of
these three species of Penicillium from insects in Korea.
Keywords Lixus imperessiventris, Meloe proscarabaeus, Muljarus japonicas, Penicillium spp.
Penicillium species are usually regarded as soil fungi [1],
but many species inhabit well-defined habitats other than
soil. They function as decomposers of dead materials and
are especially important postharvest, where they spoil
food commodities [2-5]. Some reports have shown that
Penicillium species can be isolated from insects and their
body parts [6]. In this study, we report three species of
Penicillium isolated from three different insect species in
Korea.
hypochlorite solution for 3 min, rinsed with plenty of
sterile distilled water, then dried using filter paper. Surface
sterilized cadavers were plated onto potato dextrose agar
(PDA) containing 0.25 mg/mL chloramphenicol to inhibit
bacterial growth and incubated at 25oC. Hyphae of the
fungi growing and sporulating on cadavers (and on PDA
medium) were cut, transferred to fresh PDA plates and
incubated at 25oC.
ITS sequencing analysis. Fungal genomic DNA samples
were extracted using InstaGene Matrix (Bio-Rad Laboratories,
Hercules, CA, USA). The primers ITS1 primer (5'-TCCGTAGGTGAACCTGCGG-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 primer (5'-TCCTCCGCTTATTGATATGC-3') were used for the PCR. The PCR
reaction was performed with 20 ng of genomic DNA as the
template in a 30 µL reaction mixture by using EF-Taq
(SolGent, Daejeon, Korea) as follows: activation of Taq
polymerase at 95oC for 2 min, 35 cycles of 95oC for 1 min,
o
o
55 C, and 72 C for 1 min each were performed, finishing
o
with a 10-min step at 72 C. The amplification products
were purified with a multiscreen filter plate (Millipore Corp.,
Bedford, MA, USA). Sequencing reactions were performed
using the PRISM BigDye Terminator v3.1 Cycle Sequencing
Kit. The DNA samples containing the extension products
were added to Hi-Di formamide (Applied Biosystems,
Foster City, CA, USA). The mixture was incubated at 95°C
for 5 min, followed by 5 min on ice, and then analyzed by
the ABI Prism 3730XL DNA analyzer (Applied Biosystems,
Foster City, CA, USA). The sequences were compared
using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/
Collection of insects and fungal isolation. Insect
samples were collected from preserved specimens of the
Insect Museum at Kangwon National University in
Chuncheon (Gangwon Province, Korea). Collected insects
were placed in a laboratory clean box for isolation of fungi.
Collected insects were surface sterilized in a 2% sodium
Mycobiology 2013 June, 41(2): 116-119
http://dx.doi.org/10.5941/MYCO.2013.41.2.116
pISSN 1229-8093 • eISSN 2092-9323
© The Korean Society of Mycology
*Corresponding author
E-mail: younslee@kangwon.ac.kr
Received February 4, 2013
Revised March 4, 2013
Accepted April 17, 2013
This is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted
non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
116
Three Penicillium spp. Isolated from Insects in Korea
Blast) for identification of the isolates. Sequences used to
calculate phylogeny were first determined using BLAST
results from databases [7] and a phylogenetic tree was
subsequently prepared by the neighbor-joining method [8].
Morphological characteristics and identification.
Penicillium polonicum: Conidiophores are observed as twostage branched (terverticillate) with all elements adpressed
and stipes rough-walled. Conidia were smooth, globose to
subglobose, and 3~4 µm in diameter. Morphologically,
conidia and conidiophores of our strain were similar to
those of Penicillium polonicum (Fig. 1). When the internal
transcribed spacer (ITS) sequences of the strain were
117
compared with related species retrieved from GenBank,
sequence analysis by BLAST indicated that KNU12-3-2
was highly related to P. polonicum, with a 99% sequence
similarity (Table 1, Fig. 2).
Penicillium steckii: Conidia observed were broadly
ellipsoidal. Conidiophores from surface hyphae, symmetrically
biverticillate, stipes smooth, and width 2.2~3.0 µm; metulae
in whorls of 3~6, 13~18 × 2.5~3.3 µm; ampulliform phialides,
7.0~10 × 2.2~3.0 µm; conidia smooth-walled, broadly
ellipsoidal, and slightly fusiform in some strains, 2.3~3.1 ×
2.0~2.6 µm (Fig. 3). When the ITS sequences of the strain
were compared with related species retrieved from GenBank,
sequence analysis by BLAST indicated that KNU12-1-8
Fig. 1. Morphological characteristics of Penicillium polonicum isolated from Muljarus japonicas. A, Morphology of the host
insect (M. japonicas); B, Colony on potato dextrose agar after 7 days of incubation; C, Mycelia of P. polonicum; D, Spores of P.
polonicum; E, Conidia of P. polonicum (scale bars: C~E = 10 µm).
Table 1. ITS sequence analysis for the identification of Penicillium species
Isolate No.
Putative species
Related Genbank accession No.
Identity (%)
KNU12-1-8
KNU12-2-9
KNU12-3-2
P. polonicum
P. steckii
Penicillium sp.
AF033475.1
HM469415.1
HQ832995.1
568/570 (99)
577/580 (99)
574/574 (100)
Fig. 2. Phylogenetic tree (using internal transcribed spacer sequences) showing the closest known relatives of newly reported
Penicillium species in Korea. Numbers above the branches indicate bootstrap values of distance.
118
Lamsal et al.
Fig. 3. Morphological characteristics of Penicillium steckii isolated from Meloe proscarabaeus. A, Morphology of the host insect
(M. proscarabaeus); B, Morphology of P. steckii on potato dextrose agar; C, Spores of P. steckii under a microscope; D, Mycelia
of P. steckii (scale bars: D, E = 10 µm).
Fig. 4. Morphological characteristics of Penicillium sp. isolated from Lixus imperessiventris. A, Morphology of host insect (L.
imperessiventris); B, Morphology of Penicillium sp. on potato dextrose agar; C, D, Mycelia and spores of Penicillium sp. under a
microscope (scale bars: D, E = 10 µm).
was highly related to P. steckii with 99% sequence
similarity (Table 1, Fig. 2).
Penicillium sp.: The mycelium typically consists of a highly
branched network of multinucleate, septate, and usually
colorless hyphae. Many-branched conidiophores sprout on
the mycelia, bearing individually constricted conidiospores.
The conidiospores are the main dispersal route of the
fungi, and are often greenish in color. Conidia are globose,
ellipsoidal, cylindrical or fusiform, hyaline or greenish, and
smooth or rough-walled (Fig. 4). When ITS sequences of
the strain were compared with related species retrieved
from GenBank, sequence analysis by BLAST indicated that
Three Penicillium spp. Isolated from Insects in Korea
KNU12-2-9 was highly related to Penicillium sp. with
100% sequence similarity (Table 1, Fig. 2).
ACKNOWLEDGEMENTS
This study was supported as part of a project on the
survey and excavation of Korean indigenous species from
the National Institute of Biological Resources (NIBR)
under the Ministry of Environment, Republic of Korea.
REFERENCES
1. Pitt JI. The genus Penicillium and its teleomorphic states
Eupenicillium and Talaromyces. London: Academic Press;
1979.
2. Janisiewicz WJ. Postharvest biological control of blue mold
on apples. Phytopathology 1987;77:481-5.
3. Pitt J, Hocking AD. Fungi and food spoilage. Cambridge;
119
Cambridge University Press; 1997.
4. Holmes GJ, Eckert JW. Sensitivity of Penicillium digitatum
and P. italicum to postharvest citrus fungicides in California.
Phytopathology 1999;89:716-21.
5. Morales H, Marín S, Rovira A, Ramos AJ, Sanchis V. Patulin
accumulation in apples by Penicillium expansum during
postharvest stages. Lett Appl Microbiol 2007;44:30-5.
6. Seifert KA, Hoekstra ES, Frisvad JC, Louis-Seize G. Penicillium
cecidicola, a new species on cynipid insect galls on Quercus
pacifica in the western United States. Stud Mycol 2004;50:
517-23.
7. Dereeper A, Audic S, Claverie JM, Blanc G. BLASTEXPLORER helps you building datasets for phylogenetic
analysis. BMC Evol Biol 2010;10:8.
8. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet
F, Dufayard JF, Guindon S, Lefort V, Lescot M, et al.
Phylogeny.fr: robust phylogenetic analysis for the nonspecialist. Nucleic Acids Res 2008;36:W465-9.